Anti-proliferation activity of 1,25-(OH)2vitamin D3 (D3) in prostate cancer cells highlights the therapeutic potential of D3/D3 analogs. This potential is, however, limited by D3/D3 analog-induced hypercalcemia and hypercalciurea. Regulatory molecules and pathways, which are involved in D3-mediated proliferation inhibition of prostate cancer cells, can be targeted to inhibit tumor growth, presenting opportunities for therapeutic exploitation and development of new lines of defense against prostate cancer. Since metastatic prostate cancer is often associated with elevated levels of E2F1, which is a key cell cycle regulatory transcription factor, we designed this project to identify regulatory factors which, through cross-talk with androgen receptor (AR), vitamin D3 receptor (VDR) and E2F1, are integrally linked to the anti-proliferation action of D3. In novel findings we show that androgen-regulated E2F1 induction in human prostate cancer cells is interfered by D3 due to transcriptional repression. The corepressor Alien and anti-proliferative protein prohibitin appear to play roles in the repression. AR and VDR were present at the regulated promoter during both induction and de-induction of E2F1. Transcription factors WSTF and E47 occupied regulated chromatin in response to androgen. Prohibitin occupied this region in D3- and androgen+D3-treated cells; but Alien occupancy occurred only in androgen+D3 co-treated cells. Clinically important anti-androgen Casodex induced Alien recruitment. We hypothesize a) WSTF and E47 are part of an AR-associated coactivator complex, while D3-bound VDR promotes co-regulator switch to a prohibitin- and Alien-included corepressor assembly, which mediates E2F1 gene repression and reduced cell proliferation; b) E2F1 repression is aided by self inhibition at an auto-regulated site in the E2F1 promoter that results from enhanced E2F1 interaction with Alien, prohibitin or well-known E2F1 partner Rb (retinoblastoma protein), and with Alien/prohibitin/Rb-recruited corepressors. Four Specific Aims will examine: 1), roles of Alien, prohibitin, Rb, and associated corepressors in E2F1 activity in prostate cancer cells treated with androgen or D3 or androgen+D3; 2), interplay of Alien and prohibitin with WSTF and E47 in regulating E2F1 gene expression; 3), roles of endogenous WSTF/E47/Alien/prohibitin in androgen-stimulated and D3-inhibited prostate cancer cell proliferation and in the growth of xenograft prostate tumors in immune-deficient mice; 4), additional components of the holo corepressor complex, following affinity enrichment, for roles in E2F1 gene silencing and in the inhibition of prostate cancer cell proliferation. Methods: We will use Co-IP, chromatin IP, transfection, ShRNAs, inducible protein expression, lentivirus infection, cell proliferation, xenograft tumor growth in SCID mice, protein enrichment and mass spectrometry. Significance: Knowledge of the factors responding to the androgen-mediated proliferation stimulation, and to the D3-mediated anti-proliferation signaling will define mechanisms that may be targeted to develop novel prostate cancer therapeutics. The project has high relevance to health care issues of the Veterans population. PUBLIC HEALTH RELEVANCE: Project Narrative: Prostate cancer is a major health concern among elderly men, and metastatic prostate cancer is a leading cause of cancer death in men in the USA, second only to death from lung cancer. Risk for the disease rises from the 5th decade of life. The VA demography is steadily shifting to a late-life population, with a projection that by the year 2020 about 46% of the veteran population will reach age 65 or more. Vitamin D deficiency is a common problem among the elderly. Population based research has shown that low serum vitamin D levels in men correlate with increased prostate cancer risk. In the laboratory, research involving cells and animals has shown that the active form of vitamin D (known as vitamin D3) is very effective in reducing growth and proliferation of prostate tumor cells. Indeed, research in recent years revealed that high vitamin D levels protect human body from many cancers, such as cancer of the colon, breast and prostate. However, high-dose clinically effective D3 and synthetic compounds that act like D3 (known as D3 analogs) are highly toxic. Thus they cannot be used in prostate cancer treatment. A protein known as E2F1 plays a key role in promoting the growth and multiplication of normal cells. However, high activity of E2F1 and high levels of this protein promote excessive and uncontrolled multiplication of cells in the prostate and other tissues, causing cancer. Treatment-resistant prostate cancer tissues in a large number of patients are found to contain much higher levels of E2F1 than normal prostate tissues. We have designed a study to investigate why E2F1 activity and E2F1 protein levels increase in prostate cancer cells, and how D3 can inhibit this increase. We will undertake various analyses using cutting edge research technology to find out how specific regulatory protein molecules are influenced by D3 in such a way that they silence or reduce the activity and production of E2F1. Our study is expected to find alternative ways to reduce E2F1 activity and protein levels to circumvent use of high-dose D3. Information gleaned from our research may open up new strategies for prevention and clinical management of prostate cancer.